Nitric oxide-releasing medical devices

ABSTRACT

A polymeric composition capable of releasing nitric oxide under physiological conditions which includes a biopolymer, such as a peptide, polypeptide, protein, oligonucleotide or nucleic acid, to which is bound a nitric oxide-releasing N 2 O 2   −  functional group; pharmaceutical compositions comprising the polymeric composition; and methods of treating biological disorders in which dosage with nitric oxide is therapeutic.

RELATED APPLICATION

[0001] This is a continuation-in-part of U.S. Ser. No.. 08/121,169,filed Sep. 14, 1993. The entire disclosure of the '169 application isincorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to polymeric compositions capableof releasing nitric oxide. In particular, the present invention relatesto polymeric compositions comprising a biopolymer, such as a peptide,polypeptide, protein, oligonucleotide, nucleic acid, or the like towhich is bound a nitric oxide-releasing N₂O₂ ⁻ functional group,pharmaceutical compositions comprising such polymeric compositions, andmethods of treating biological disorders with such a biopolymericcomposition.

BACKGROUND OF THE INVENTION

[0003] Nitric oxide (NO) has recently been implicated in a variety ofbioregulatory processes, including normal physiological control of bloodpressure, macrophage-induced induced cytostasis and cytotoxicity, andneurotransmission (Moncada et al., “Nitric Oxide from L-Arginine: ABioregulatory System,” Excerota Medica, International Congress Series897 (Elsevier Science Publishers B. V.: Amsterdam, 1990); Marletta etal., “Unraveling the Biological Significance of Nitric Oxide,”Biofactors, 2, 219-225 (1990); Ignarro, “Nitric Oxide. A Novel SignalTransduction Mechanism for Transcellular Communication,” Hypertension(Dallas), 16, 477-483 (1990)). A number of compounds have been developedwhich are capable of delivering nitric oxide, including compounds whichrelease nitric oxide upon being metabolized and compounds which releasenitric oxide spontaneously in aqueous solution.

[0004] Those compounds which release nitric oxide upon being metabolizedinclude the widely used nitrovasodilators glyceryl trinitrate and sodiumnitroprusside (Ignarro et al., J. Pharmacol. Exp. Ther., 218, 739-749(1981); Ignarro, Annu. Rev. Pharmacol. Toxicol., 30, 535-560 (1990);Kruszyna et al., Toxicol. Appl. Pharmacol., 91, 429-438 (1987); Wilcoxet al., Chem. Res. Toxicol., 3, 71-76 (1990). Another compound,S-nitroso-N-acetylpenicillamine, has been reported to release nitricoxide in solution and to be effective at inhibiting DNA synthesis (Garget al., Biochem. and Biophys. Res. Comm., 171, 474-479 (199.0)).

[0005] Numerous nitric oxide-nucleophile complexes have been described,e.g., Drago, ACS Adv. Chem. Ser., 36, 143-149 (1962). See also Longhiand Drago, Inog. Chem., 2, 85 (1963). Some of these complexes are knownto evolve nitric oxide on heating or hydrolysis, e.g., Maragos et al.,J. Med. Chem. 34, 3242-3247 (1991).

[0006] The cytostatic effect of nitric oxide solutions on tumor cells invitro has been demonstrated. In particular, it has been shown thatsolutions of nitric oxide inhibit DNA synthesis and mitochondrialrespiration of tumor cells in vitro (Hibbs et al., Biochem. and Bioahys.Res. Comm., 157, 87-94 (1988); Stuehr et al., J. Exp. Med., 169,1543-1555 (1989)).

[0007] Endothelium-derived relaxing factor (EDRF) is a labile humoralagent which is part of a cascade of interacting agents involved in therelaxation of vascular smooth muscle. EDRF is thus important in thecontrol of vascular resistance to blood flow and in the control of bloodpressure. Some vasodilators act by causing EDRF to be released fromendothelial cells. (See Furchgott, Ann. Rev. Pharmacol. Toxicol., 24,175-197 (1984).) In 1987, Palmer et al., presented evidence that EDRF isidentical to the simple molecule, nitric oxide, NO (Nature, 317, 524-526(1987)), though more recently,.that conclusion has been challenged(Myers et al., Nature, 345, 161-163, 1990)).

[0008] Nitric oxide in its pure form, however, is a highly reactive gashaving limited solubility in aqueous media (WHO Task Group onEnvironmental Health Criteria for Oxides of Nitrogen, Oxides ofNitrogen, Environmental Health Criteria 4 (World Health Organization:Geneva, 1977)). Nitric oxide, therefore, is difficult to introducereliably into most biological systems without premature decomposition.

[0009] The difficulty in administering nitric oxide can be overcome insome cases by administering nitric oxide pharmacologically in prodrugform. The compounds glyceryl trinitrate and sodium nitroprusside arerelatively stable and release nitric oxide only on activation (Ignarroet al., J. Pharmacol. ExR. Ther., 218, 739-749 (1981); Ignarro, Annu.Rev. Pharmacol. Toxicol., 30, 535-560 (1990); Kruszyna et al., Toxicol.Arol. Pharmacol., 91, 429-438 (1987); Wilcox et al., Chem. Res.Toxicol., 3, 71-76 (1990)). While this feature may be an advantage insome applications, it can also be a significant liability, as in thedevelopment of tolerance to glyceryl trinitrate via the exhaustion ofthe-relevant enzyme/cofactor system (Ignarro et al., Annu. Rev.Pharmacol. Toxicol., 25, 171-191 (1985); Kuhn et al., J. Cardiovasc.Pharmacol., 14 (Suppl. 11), S47-S54 (1989)) and toxicity frommetabolically produced cyanide during prolonged administration ofnitroprusside (Smith et al., “A Potpourri of Biologically ReactiveIntermediates” in Biological Reactive Intermediates IV. Molecular andCellular Effects and Their Impact on Human Health (Witmer et al., eds.),Advances in Experimental Medicine and Biology Volume 283 (Plenum Press:New York, 1991), pp. 365-369).

[0010] Evidence that nitric oxide is released from the endothelial cellsand is responsible for the relaxation of the vascular smooth muscle, andhence the control of blood pressure, has resulted in the development ofartificial agents that can deliver nitric oxide in vivo. A veryimportant class of such agents is the nitric oxide-nucleophilecomplexes. Recently, a method for treating cardiovascular disorders in amammal with certain nitric oxide-nucleophile complexes was disclosed,e.g. in U.S. Pat. No. 4,954,526. These compounds contain the anionicN₂O₂ ⁻ group or derivatives thereof. See also, Maragos et al., J. Med.Chem., 34, 3242-3247 (1991). Many of these compounds have provenespecially promising pharmacologically because, unlike nitrovasodilatorssuch as nitroprusside and nitroglycerin, they release nitric oxidewithout first having to be activated. The only other series of drugscurrently known to be capable of releasing nitric oxide purelyspontaneously is the S-nitrosothiol series, compounds of structureR—S—NO (Stamler et al., Proc. Natl. Acad. Sci. U.S.A., 89, 444-448(1992); Stamler et al., Proc. Natl. Acad. Sci. U.S.A., 89, 8087-8091(1992)); however, the R—S—NO—NO reaction is kinetically complicated anddifficult to control (Morley et al., J. Cardiovasc. Pharmacol., 21,670-676 (1993)). The N₂O₂ ⁻ containing compounds are thus advantageousamong drugs currently known in that they decompose at any given pH via acleanly first order reaction to provide doses of nitric oxide that canbe predicted, quantified, and controlled. See, e.g., Maragos et al., J.Med. Chem., 34, 3242-3247 (1991).

[0011] Nitric oxide/nucleophile complexes which release nitric oxide inaqueous solution are also disclosed in U.S. Pat. Nos. 5,039,705,5,185,376, 5,155,137, 5,208,233, 5,212,204, 5,250,550 and 5,366,997(issue date 11/22/94) as well as in pending U.S. patent applicationsSer. No. 07/764,908 (filed Sep. 24, 1991), 07/858,885 (filed Mar. 27,1992), 07/867,759 (filed Apr. 13, 1992), 07/935,565 (filed Aug. 24,1992), 08/017,270 (filed Feb. 12, 1993), and 08/121,169 (filed Sep. 14,1993) as useful therapeutic agents (see also Maragos et al., J. Med.Chem., 34, 3242-3247 (1991)).

[0012] Despite the promise of the nitric oxide/nucleophile adducts thathave been investigated, their pharmacological application has beenlimited by their tendency to distribute evenly throughout the medium.Such even distribution is a great advantage in many researchapplications, but tends to compromise their selectivity of action.Another limitation to the application of these nitric oxide/nucleophileadducts is their propensity for relatively rapid release of nitric oxidewhich may necessitate frequent dosing to achieve a prolonged biologicaleffect. Thus there remains a need for nitric oxide-releasingcompositions which are capable of concentrating the effect of the nitricoxide release to a situs of application and for which nitric oxiderelease may be controlled for effective dosing.

[0013] It is, therefore, a principal object of the present invention toprovide a polymeric composition comprising a biopolymer to which isbound a N₂O₂ ⁻ functional group and which is capable of releasing NOunder physiological conditions. Another object of the invention is toprovide a polymeric composition comprising a biopolymer to which isbound a N₂O₂ ⁻ functional group whose release of NO can be controlledsuch that local or cell/tissue specific release can be effected. It isanother object of the present invention to provide a polymericcomposition comprising a biopolymer to which is bound a N₂O₂ ⁻functional group whose release of NO is such that a prolonged biologicaleffect can be attained. Yet another object of the present invention isto provide pharmaceutical compositions comprising such biopolymericcompositions. It is also an object of the present invention to provide amethod of treating a biological disorder involving the administration ofsuch biopolymeric compositions. These and other objects and advantagesof the present invention, as well as additional inventive features, willbe apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

[0014] The present invention provides a polymeric composition capable ofspontaneously releasing nitric oxide under physiological conditions. Thepolymeric composition comprises a biopolymer to which is bound a nitricoxide-releasing N₂O₂ ⁻ functional group. “Biopolymer(ic)” is meant toinclude any biological polymer, such as peptides, polypeptides,proteins, oligonucleotides, and nucleic acids, including those thatcontain naturally occurring and/or nonnaturally occurring subunits.Specific examples include antibodies or fragments thereof and peptidehormones, proteins, and growth factors for which the target cell typehas a high population of receptors. The preferred nitric oxide-releasingN₂O₂ ⁻ functional group which is used to form the biopolymer-boundNONOates of the present invention is defined by the formula:

[0015] wherein X is an organic or inorganic moiety and X′ may be thesame as X, or it may be a pharmaceutically acceptable metal center, apharmaceutically acceptable cation, or the like. The N₂O₂ ⁻ group isbonded to the biopolymer through either or both the linking groups X andX′.

[0016] By “bound to a polymer,” it is meant that the N₂O₂ ⁻ functionalgroup is associated with, part of, incorporated with or contained withinthe biopolymer physically or chemically. Bonding of the N₂O₂ ⁻functional group to the polymer can be achieved by covalent bonding ofthe N₂O₂ ⁻ group to the biopolymer through a linking group X or X′.Chemical bonding of the N₂O₂ ⁻ functional group to the biopolymer may beby, for example, covalent bonding of the linking group X or X′ to thebiopolymer such that the linking group forms part of the biopolymeritself, i.e., is in the biopolymer backbone or is attached to pendantgroups on the biopolymer backbone. The manner in which the nitricoxide-releasing N₂O₂ ⁻ functional group is associated with, part of,incorporated with or contained within, i.e., “bound,” to the polymer isinconsequential to the present invention and all means of association,incorporation and bonding are contemplated herein.

[0017] In another aspect of the invention, the biopolymer-bound nitricoxide-releasing compositions of the present invention can be bound to orphysically associated with polymers that are not biopolymers (referredto hereinafter as “non-biopolymers”).

[0018] The present invention also provides a pharmaceutical compositionwhich includes a pharmaceutically acceptable carrier and a polymericcomposition comprising a biopolymer to which is bound a nitricoxide-releasing N₂O₂ ⁻ functional group.

[0019] The invention further provides a method of treating biologicaldisorders in which dosage with nitric oxide would be therapeutic whichcomprises administering to a mammal afflicted with such a biologicaldisorder a polymeric composition, comprising a biopolymer to which isbound a nitric oxide-releasing N₂O₂ ⁻ functional group, in-an amountsufficient to release a therapeutically effective amount of nitricoxide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention is predicated on the discovery that usefulpharmacological agents can be provided by incorporating nitricoxide-releasing N₂O₂ ⁻ functional groups into a biopolymer. Accordingly,the N₂O₂ ⁻ functional group is “bound to the polymer” as that term hasbeen defined herein. The term NONOate is used herein as a shorthand torefer to the nitric oxide-releasing N₂O₂ ⁻ group.

[0021] It has been discovered that incorporation of a NONOate into abiopolymer provides a biopolymer-bound NONOate composition that can beapplied with specificity to a biological site of interest. Site specificapplication of the biopolymer-bound NONoate enhances the selectivity ofaction of the nitric oxide-releasing NONOate. If N₂O₂ ⁻ functionalgroups attached to the biopolymer are necessarily localized, then theeffect of their nitric oxide release will be concentrated in the tissueswith which they are in contact. If the biopolymer is soluble,selectivity of action can still be arranged, for example, by attachmentto or derivatization of an antibody specific to the target tissue.Similarly, attachment of N₂O₂ ⁻ groups to small peptides that mimic therecognition sequences of ligands for important receptors provideslocalized concentrated effect of nitric oxide release, as wouldattachment to oligonucleotides capable of site-specific interactionswith target sequences in a nucleic acid. Other proteins, peptides,polypeptides, nucleic acids and polysaccharides, including hormones andmotility, chemotactic and extravasating factors or agents, can besimilarly utilized.

[0022] By way of illustration, a piperazine monoNONOate derivative canbe covalently attached to a polypeptide containing the IKVAV recognitionsequence important in tumor cell chemotaxis. Through retention of boththe capacity to regenerate NO as an antichemotactic agent and theaffinity of the IKVAV sequence for tumor cells and/or sites in thevascular and lymphatic systems where the tumor cells tend to attach,metastasis can be reduced or even prevented.

[0023] While not being bound to any particular theory, it is believedthat longevity of nitric oxide release in the biopolymer-bound NONOatecompositions of the present invention is to be attributed both to thephysical structure of the composition and to electrostatic effects.Thus, it is believed that if the biopolymer is an insoluble solid, N₂O₂⁻ groups near the surface of the particle should be available for rapidrelease while those that are more deeply imbedded are stericallyshielded, requiring more time and/or energy for the nitric oxide to workits way into the medium. Unexpectedly, it has been found that increasingpositive charge in the vicinity of an N₂O₂ ⁻ functional group also tendsto increase the halflife of nitric oxide generation. The mechanism ofthis rate retardation may be attributable simply to repulsiveelectrostatic interactions, i.e., increasing the number of H+− repellingpositive charges in the vicinity of the N₂O₂ ⁻ groups inhibits attack ofpositively charged H⁺ ions on the N₂O₂ ⁻ functional group and slows therate of its H⁺⁻ catalyzed decomposition. For example, by attaching aminogroups to the polymeric support that are capable of forming the nitricoxide-releasing N₂O₂ ⁻ functional group on reaction with nitric oxide,partially converted structures can be produced on less-than-exhaustivetreatment with nitric oxide that after exposure to water contain a largenumber of positively charged ammonium centers surrounding the N₂O₂ ⁻group that electrostatically inhibit the approach of H⁺ ions capable ofinitiating nitric oxide loss from the nitric oxide-releasing N₂O₂ ⁻functional group.

[0024] The nitric oxide-releasing N₂O₂ ⁻ functional groups that arebound to the biopolymer generally are capable of releasing nitric oxidein an aqueous environment spontaneously upon contacting an aqueousenvironment, i.e., they do not require activation through a redoxreaction or electron transfer such as is required for glyceryltrinitrate and sodium nitroprusside. Some of the nitricoxide/nucleophile complexes useful in the context of the presentinvention do require activation by particular means, but only asnecessary to free the nitric oxide-releasing X[N(O)NO]⁻ group in thevicinity of the particular cells of interest. As an example, covalentattachment of a protecting group to the anionic [N(O)NO]⁻ functionprovides a means of postponing nitric oxide release until the moleculereaches an organ capable of metabolically removing the protecting group.By choosing a protecting group that is selectively cleaved by enzymesspecific to a tumor, biological disorder, cell, or tissue of interest,for example, the action of the nitric oxide/nucleophile complex can betargeted to maximize the desired effect. While the biopolymer-boundNONOate compositions of the present invention are capable of releasingnitric oxide in an aqueous solution, such a compound preferably releasesnitric oxide under physiological conditions.

[0025] For example, a NONOate functionality can be attached to atumor-specific antibody or other protein which has one or more lysineside chain amino groups that are unnecessary to the function of theprotein by reacting said lysine group(s) with a derivatizing agentcapable of covalently attaching first to the lysine amino nitrogen thenin a subsequent step to the sulfur atom of an O-functionalized NONOatecontaining a free thiol grouping elsewhere in the molecule. Once such aprotein arrives at the desired target tissue after systemic application,enzymatic or hydrolytic removal of the substituent bound to oxygen freesthe anionic NONOate function to concentrate NO release at that site.

[0026] The preferred nitric oxide-releasing N₂O₂ ⁻ functional groupwhich is used to form the biopolymer-bound NONOates of the presentinvention is defined by the formula:

[0027] wherein X is an organic or inorganic moiety and X′ is an organicor inorganic substituent, a pharmaceutically acceptable metal center, apharmaceutically acceptable cation, or the like. The N₂O₂ ⁻ group isbonded to the biopolymer through either or both the linking groups X andX′.

[0028] The nitric oxide-releasing N₂O₂ ⁻ functional group is preferablya nitric oxide/nucleophile adduct, e.g., a complex of nitric oxide and anucleophile; most preferably a nitric oxide/nucleophile complex whichcontains the anionic moiety X[N(O)NO]⁻, where X is any suitablenucleophile residue. The nucleophile residue is preferably that of aprimary amine (e.g., X=(CH₃)₂CHNH, as in (CH₃)₂CHNH[N(O)NO]Na), asecondary amine (e.g., X=(CH₃CH₂)₂N, as in (CH₃CH₂)₂N[N(O)NO]Na), apolyamine (e.g., X=spermine, as in the zwitterion H₂N(CH₂)₃NH₂⁺(CH₂)₄N[N(O)NO]⁻(CH₂)₃NH₂, X=2-(ethylamino)ethylamine, as in thezwitterion CH₃CH₂N[N(O)NO]⁻CH₂CH₂NH₃ ⁺, orX=3-(n-propylamino)propylamine, as in the zwitterionCH₃CH₂CH₂N[N(O)NO]⁻CH₂CH₂CH₂NH₃ ⁺), or oxide (i.e., X═O⁻, as inNaO[N(O)NO]Na), or a derivative thereof. Such nitric oxide/nucleophilecomplexes are capable of delivering nitric oxide in a biologicallyusable form as a predictable rate.

[0029] Other suitable nitric oxide/nucleophile complexes include thosehaving the following formulas:

[0030] wherein J is an organic or inorganic moiety, M^(+x) is apharmaceutically acceptable cation, where x is the valence of thecation, a is an integer of at least one, and b and c are the smallestintegers that result in a neutral compound, as described in U.S. Pat.No. 5,208,233, and incorporated herein by reference;

[0031] wherein b and d are the same or different and may be zero or one,R₁, R₂, R₃, R₄, and R₅ are the same or different and may be hydrogen,C₃₋₈ cycloalkyl, C₁₋₁₂ straight or branched chain alkyl, benzyl,benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl,or 2,2,2-trichloro-t-butoxycarbonyl, and x, y, and z are the same ordifferent and are integers from 2 to 12, as described in U.S. Pat. No.5,155,137, incorporated herein by reference;

[0032] and R₇ are the same or different and may be hydrogen, C₃₋₈cycloalkyl, C₁₋₁₂ straight or branched chain alkyl, benzyl, benzoyl,phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl, or2,2,2-trichloro-t-butoxycarbonyl, f is an integer from 0 to 12, with theproviso that when B is the substituted piperazine moiety

[0033] then f is an integer from 2 to 12, as described in U.S. Pat. No.5,155,137, incorporated herein by reference;

[0034] wherein R₈ is hydrogen, C₃₋₈ cycloalkyl, C₁₋₁₂ straight orbranched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl,trifluoroacetyl, p-toluyl, t-butoxycarbonyl, or2,2,2-trichloro-t-butoxycarbonyl, R₉ is hydrogen or a C₁-C₁₂ straight orbranched chain alkyl, and g is 2 to 6, as described in U.S. Pat. No.5,250,550, incorporated herein by reference;

[0035] wherein R₁ and R₂ are independently selected from the groupconsisting of a straight chain or branched chain C₁-C₁₂ alkyl group anda benzyl group, or else R₁ and R₂, together with the nitrogen atom, arebonded to form a heterocyclic group, preferably a pyrrolidino,piperidino, piperazino or morpholino group, M^(+x) is a pharmaceuticallyacceptable cation, and x is the valence of the cation, as described inU.S. Pat. Nos. 5,039,705 and 5,208,233 and U.S. patent application Ser.No. 08/017,270, filed Feb. 12, 1993, and incorporated here in byreference;

K[(M)^(x′) _(x)(L)_(y)(R¹R²N—N₂O₂)_(z)]  (VI)

[0036] wherein M is a pharmaceutically acceptable metal, or, where x isat least two, a mixture of two different pharmaceutically acceptablemetals, L is a ligand different from (R¹R²N—N₂O₂) and is bound to atleast one metal, R¹ and R² are each organic moieties and may be the sameor different, x is an integer of from 1 to 10, x′ is the formaloxidation state of the metal M, and is an integer of from 1 to 6, y isan integer of from 1 to 18, and where y is at least 2, the ligands L maybe the same or different, z is an integer of from 1 to 20, and K is apharmaceutically acceptable counterion to render the compound neutral tothe extent necessary, as described in U.S. patent application Ser. No.07/858,885, filed Mar. 27, 1992, and incorporated herein by reference;

[R—N(H)N(NO)O—]_(y)X  (VII)

[0037] wherein R is C₂₋₈ lower alkyl, phenyl, benzyl, or C₃₋₈cycoloalkyl, any of which R groups may be substituted by one to threesubstituents, which are the same or different, selected from the groupconsisting of halo, hydroxy, C₁₋₈ alkoxy, —NH₂, —C(O)NH₂, —CH(O),—C(O)OH, and —NO₂, X is a pharmaceutically acceptable cation, apharmaceutically acceptable metal center, or a pharmaceuticallyacceptable organic group selected from the group consisting of C₁₋₈lower alkyl, —C(O)CH₃, and —C(O)NH₂, and y is one to three, consistentwith the valence of X, as described in U.S. Pat. No. 4,954,526 andincorporated herein by reference; and

[0038] wherein R₁ and R₂ are independently chosen from C₁₋₁₂ straightchain alkyl, C₁₋₁₂ alkoxy or acyloxy substituted straight chain alkyl,C₂₋₁₂ hydroxy or halo substituted straight chain alkyl, C₃₋₁₂ branchedchain alkyl, C₃₋₁₂ hydroxy, halo, alkoxy, or acyloxy substitutedbranched chain alkyl, C₃₋₁₂ straight chain olefinic and C₃₋₁₂ branchedchain olefinic which are unsubstituted or substituted with hydroxy,alkoxy, acyloxy, halo or benzyl, or R₁ and R₂ together with the nitrogenatom to which they are bonded form a heterocyclic group, preferably apyrrolidino, piperidino, piperazino or morpholino group, and R₃ is agroup selected from C₁₋₁₂ straight chain and C₃₋₁₂ branched chain alkylwhich are unsubstituted or substituted by hydroxy, halo, acyloxy oralkoxy, C₂₋₁₂ straight chain or C₃₋₁₂ branched chain olefinic which areunsubstituted or substituted by halo, alkoxy, acyloxcy or hydroxy,C-₁₋₁₂ unsubstituted or substituted acyl, sulfonyl and carboxamido; orR₃ is a group of the formula —(CH₂)_(n)—ON═N(O)NR₁R₂, wherein n is aninteger of 2-8, and R₁ and R₂ are as defined above; with the provisothat R₁, R₂ and R₃ do not contain a halo or a hydroxy substituent α to aheteroatom, as described in U.S. application Ser. No. 07/950,637, filedSep. 23, 1992.

[0039] Any of a wide variety of biopolymers can be used in the contextof the present invention. Biopolymers suitable for use include peptides,polypeptides, proteins, oligonucleotides, nucleic acids, e.g., RNA andDNA, antibodies, peptide hormones, glycoproteins, glycogen, and thelike. Alternatively, a subunit of a biopolymer, such as a fatty acid,glucose, an amino acid, a succinate, a ribonucleotide, a ribonucleoside,a deoxyribonucleotide, and a deoxyribonucleoside can be used.Illustrative examples include antibodies or fragments thereof;extracellular matrix proteins such as laminin, fibronectin, or theircell attachment-site peptide recognition sequences, such as RGDS, IKVAV,YIGSR, and the like; and growth factors, peptide hormones, and otherpolypeptides for which there are high-affinity cell surface receptorsites, such as EGF, TGFα, TGFβ and TNF. Such molecules, upon receptorbinding, may be internalized into the target cells, thereby facilitatingintracellular delivery of the NO donor moiety.

[0040] The nitric oxide-releasing N₂O₂ ⁻ functional groups may be boundto the biopolymer by formation of a nitric oxide/nucleophile complex ofthe type and having the formulas of those described above, in situ onthe biopolymer. The N₂O₂ ⁻ functional group may be attached to an atomin the backbone of the biopolymer, or it may be attached to a grouppendant to the biopolymer backbone, or it may simply be entrapped in thebiopolymer matrix. Where the N₂O₂ ⁻ functional group is attached to thebiopolymer backbone, the biopolymer includes in its backbone sites whichare capable of reacting with nitric oxide to bind the nitric oxide forfuture release. For example, the biopolymer can include nucleophilicnitrogen atoms which react with nitric oxide to form the N₂O₂ ⁻functional group at the nitrogen in the backbone. Where the N₂O₂ ⁻functional group is a group pendant to the polymer backbone, thebiopolymer contains, or is derivatized with, a suitable pendantnucleophile residue capable of reacting with nitric oxide to form theN₂O₂ ⁻ functionality. Reaction of the biopolymer which contains asuitable nucleophilic residue, or of the suitably derivatizedbiopolymer, with nitric oxide thus provides a biopolymer-bound nitricoxide-releasing N₂O₂ ⁻ functional group.

[0041] To form the biopolymer-bound nitric oxide releasing N₂O₂ ⁻functional group, it is generally preferred to impart a net charge tothe polymer near the site on the biopolymer where the N₂O₂ ⁻ functionalgroup is to be formed. By way of illustration, several general means areavailable for synthesizing a biopolymeric composition comprising abiopolymer to which is attached a NONOate functional group. As oneexample, an ion of structure X—N₂O₂ ⁻ is reacted with an electrophilicagent (an [X′]⁺-donor) to generate a covalently bonded NONOate offormula X═N(O)═NOX′; this protected complex is then attached to thedesired biopolymer via the nucleophile residue, X, or the electrophileresidue, X′. Alternatively, a nucleophile residue that is already partof (or that can be attached to) the biopolymer can be reacted with NOunder basic conditions to give a nitric oxide complex containing a N₂O₂⁻ functional group. As a specific example, a simple amino acid bearing asecondary amino group can be reacted with nitric oxide to generate acompound in accordance with the present invention. Similarly, theNONOate functionality can be attached to a basic nitrogen in a peptide.Alternative means can be used to attach NONOate-containing molecules tothiol or activated carboxylic acid groups in a peptide, polypeptide orprotein in accordance with the present invention.

[0042] Further, by way of illustration, the N₂O₂ ⁻ functional group maybe attached to a peptide such as arg-gly-asp (RGD), to prepare themolecule arg-gly-asp-[N(O)NO]⁻. Preferably, the RGD tripeptide would beattached to the NONOate through a linking group such as additionalpeptide units. Other receptor/ligand recognition sequences may be usedanalogously.

[0043] The biopolymer-bound nitric oxide-releasing compositions of thepresent invention will find utility in a wide variety of applicationsand in a wide variety of forms depending on the biological disorder tobe treated. For example, the biopolymer-bound NONOate may itself bestructurally sufficient to serve as implants, patches, stents,liposomes, microparticles, microspheres, beads, powders, liquids, gels,monolithic resins, disks, or the like, or the biopolymer-bound NONOatecan be attached to a non-biopolymer, or the like, suitable for suchpurpose. The term non-biopolymer is used herein to mean any polymer thatis not a biopolymer. Further, by way of illustration, thebiopolymer-bound NONOate composition can be incorporated into otherpolymer matrices, substrates or the like, or it may bemicroencapsulated, or the like.

[0044] The biopolymer-bound nitric oxide/nucleophile compositions of thepresent invention have a wide range of biological utility. In view ofthe growing awareness that nitric oxide is an especially versatile andimportant bioeffector species, having been implicated mechanistically insuch critical bodily functions as vasorelaxation, neurotransmission andthe immunological response (Moncada et al., Pharmacol. Rev., 43, 109-142(1991), the compositions of the present invention find utility inapplications where nitric oxide release is needed. For example, thebiopolymer-bound NONOates may be used to reduce the risk of restenosisafter angioplasty.

[0045] The following are further illustrative of, and not in any way inlimitation of, the broad uses and applications of the biopolymer-boundcompositions of this invention. Thus, for example, in view of dramaticbut short-lived pulmonary vaso- and bronchodilatory properties exhibitedby nitric oxide (Roberts et al., Circulation (Suppl. II), 84, A1279(1991)), administration of biopolymer-bound nitric oxide/nucleophileadduct compositions into the lungs in aerosolized form may be used intreating a variety of pulmonary disorders. Since natural, endogenousnitric oxide has been identified as an effector of penile erection(Blakeslee, New York Times, Jan. 9, 1992, page A1), the biopolymer-boundnitric oxide/nucleophile adduct compositions of the present inventionmay be incorporated into suitable penile implants, preparations fortransurethral injection, dermal patches or condoms for treatment ofimpotence in men. The ability of certain monomeric nitricoxide/nucleophile adducts to inhibit platelet aggregation coupled withtheir demonstrated cytostatic activity allows for an invaluabletwo-pronged approach to prevention of restenosis following angioplasty;stents fabricated with polymer-bound nitric oxide-releasing N₂O₂ ⁻functional group compositions may be used both to inhibit cell divisionin areas with damaged endothelium and to prevent adhesion of plateletsat these locations as well, minimizing the risk of recurring blockage.With an inverse relationship between generation of nitric oxide by tumorcells and their metastatic potential having been proposed (Radomski etal., Cancer Res., 51, 6073-6078 (1991), polymer-bound nitricoxide/nucleophile compositions can be used to reduce the risk ofmetastasis in cancer patients. Similarly, it is contemplated that thebiopolymer-bound nitric oxide-releasing compositions of the presentinvention can be used to coat prostheses and medical implants, such asbreast implants, prior to surgical connection to the body as a means ofreducing the risk of solid state carcinogenesis associated therewith.Tumor-specific antibodies containing the N₂O₂ ⁻ functional group can beused to sensitize cancer cells to radiotherapy. The N₂O₂ ⁻ functionalgroup can be attached to hormones that concentrate in the uterus, wherethe release of NO can halt premature labor. With nitric oxide beingadditionally implicated in gastric motility, neurotransmission,nociception, and other natural roles, the compositions of this inventioncan be used for those applications as well.

[0046] In another aspect of the invention, there is provided apolymer-bound nitric oxide-releasing composition which comprises thenovel biopolymer-bound nitric oxide-releasing compositions of thepresent invention and a-non-biopolymer as disclosed in copendingapplication Ser. No. 07/935,565. In accordance with this aspect of theinvention, the biopolymer-bound NONOates described herein areincorporated into or bonded to a non-biopolymer. For this use, any of awide variety of polymers can be used. It is only necessary that thepolymer selected is biologically acceptable. Illustrative of polymerssuitable for use in the present invention are polyolefins, such aspolystyrene, polypropylene, polyethylene, polytetrafluorethylene,polyvinylidene difluoride, and polyvinylchloride, polyethylenimine orderivatives thereof, polyethers such as polyethyleneglycol andpolysaccharides, polyesters such as poly(lactide/glycolide), polyamidessuch as nylon, polyurethanes, colestipol and derivatives thereof. Thebiopolymeric nitric oxide-releasing compositions described above may bebound to a non-biopolymer support in a number of different ways. Forexample, the biopolymer-bound NONOates may be bound to thenon-biopolymer by coprecipitation of the biopolymer with thenon-biopolymer. Coprecipitation involves, for example, solubilizing boththe non-biopolymer and the biopolymer-bound NONOate and evaporating thesolvent. Alternatively, the biopolymer-bound NONOates can be chemicallybonded to the non-biopolymer.

[0047] The physical and structural characteristics of thenon-biopolymers suitable for use in the present invention are notnarrowly critical, but rather will depend on the end use application. Itwill be appreciated by those skilled in the art that where the resultingpolymeric composition is intended for topical, dermal, percutaneous, orsimilar use, it need not be biodegradable. For some uses, such asingestion or the like, it may be desirable that the non-biopolymerslowly dissolve in a physiological environment or that it isbiodegradable. The resulting polymeric forms can be bioerodible, durableor instantly soluble in physiological fluids.

[0048] One skilled in the art will appreciate that suitable methods ofadministering the biopolymer-bound nitric oxide-releasing N₂O₂ ⁻functional group compositions of the present invention to an animal areavailable, and, although more than one route can be used to administer aparticular composition, a particular route can provide a more immediateand more effective reaction than another route. Pharmaceuticallyacceptable carriers are also well-known to those who are skilled in theart. The choice of carrier will be determined in part by the particularcomposition, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of the pharmaceutical composition of the present invention.

[0049] Formulations suitable for oral administration can consist of (a)liquid solutions, such as an effective amount of the biopolymer-boundcomposition dissolved in diluents, such as water or saline, (b)capsules, sachets or tablets, each containing a predetermined-amount ofthe active ingredient, as solids or granules, (c) suspensions in anappropriate liquid, and (d) suitable emulsions. Tablet forms can includeone or more of lactose, mannitol, corn starch, potato starch,microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide,croscarmellose sodium, talc, magnesium stearate, stearic acid, and otherexcipients, colorants, diluents, buffering agents, moistening agents,preservatives, flavoring agents, and pharmacologically compatiblecarriers. Lozenge forms can comprise the active ingredient in a flavor,usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin or sucrose and acacia emulsions, gels, and the like containing,in addition to the active ingredient, such carriers as are known in theart.

[0050] The biopolymer-bound nitric oxide-releasing compositions of thepresent invention, alone or in combination with other suitablecomponents, can be made into aerosol formulations to be administered viainhalation. These aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like.

[0051] Formulations suitable for parenteral administration includeaqueous and non-aqueous, isotonic sterile injection solutions, which cancontain anti-oxidants, buffers, bacteriostats, and solutes that renderthe formulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

[0052] The dose administered to an animal, particularly a human, in thecontext of the present invention should be sufficient to effect atherapeutic response in the animal over a reasonable time frame. Thedose will be determined by the strength of the particular compositionsemployed and the condition of the animal, as well as the body weight ofthe animal to be treated. The size of the dose also will be determinedby the existence, nature, and extent of any adverse side-effects thatmight accompany the administration of a particular composition.

[0053] The following examples further illustrate the present invention,but do not limit the scope thereof.

[0054] In the Examples, chemiluminescence analysis for total recoverablenitric oxide from polymers containing the nitric oxide-releasing N₂O₂ ⁻functional group by acid treatment was carried out as follows:

[0055] The analysis of NO adducts, i.e., polymers containing the nitricoxide-releasing N₂O₂ ⁻ functional group, was done on a nitric oxideanalyzer and was patterned after the procedure of Maragos et al., J.Med. Chem., 34, 3242-3247 (1991). A reactor vessel fitted with a septumwas charged with a small aliquot of the polymer to be studied and thesystem was purged with helium for several minutes to remove traces ofoxygen. Two milliliters of 10 mM sulfuric acid was added by injectionthrough the septum to begin reaction. Gaseous effluent was sweptcontinuously via a fritted glass bubbler positioned at the bottom of thereactor vessel (i.e., immersed in the acid solution) into achemiluminescence detector (Thermal Energy Analyzer Model 502LC,Thermedics, Inc., Woburn, Mass.). The area of the resultingchemiluminescence signal versus time curve was electronically computedand compared with that of a known quantity of nitric oxide gas standardto determine the amount of nitric oxide produced by acid treatment ofthe polymer aliquot.

[0056] This procedure was used to estimate the total amount of nitricoxide recoverable from the polymer. To estimate the rate of nitric oxidegeneration under physiological conditions, the inventive polymers weresubjected to a procedure identical to that described above except that 2ml of 10 mM phosphate buffer, pH 7.4, at 37° C. was injected into thereactor vessel in place of the sulfuric acid solution to start thereaction.

EXAMPLES

[0057] The preparation and characterization of biopolymers containingthe nitric oxide-releasing N₂O₂ ⁻ functional group are illustrated inthe following examples:

Example I

[0058] This example illustrates the preparation of1-(2S-carboxypyrrolidin-1-yl)-1-oxo-2-hydroxydiazene, disodium salt, asshown schematically as follows:

[0059] A solution of 10 g (0.087 mol) of L-proline in 39 ml (0.18 mol)of 25% sodium methoxide in methanol and 20 ml of methanol was degassedand exposed to 40 psi of NO for 20 h. The pressure was released and thesolid residue was collected by filtration, washed with ether, and driedunder vacuum to give 17 g of a white solid: UV (0.01 M NaOH) λ_(max) (ε)250 nm (ε=4.9 mM⁻¹ cm⁻¹); NMR (D₂O) δ1.71 (m, 1 H), 1.91 (m, 2 H), 2.27(,m, 1 H), 3.27-3.43 (m, 2 H), 4.04 (m, 1 H). A methanol peak was alsopresent, but the solid was free of both proline and N-nitrosoproline.

Example II

[0060] This example illustrates the preparation of1-hydroxy-2-oxo-3-carboxymethyl-3-methyl-1-triazene, disodium salt, asshown schematically as follows:

[0061] To a solution of 8 g (0.2 mol) of sodium hydroxide in 100 ml ofmethanol and 20 ml of water was added 8.9 g (0.1 mol) of sarcosine. Thesolution was charged with 40 psi of NO and stirred at 250° C. for 48 h.The pressure was released, and the solution was evaporated in vacuo togive a white solid: UV λ_(max) 250 nm. The distillate had a strong amineodor, which was determined to be methylamine on derivatization withbenzoyl chloride.

[0062] The solid residue was dried under high vacuum, then analyzed byNMR in D₂O. Five products were detected by NMR: methylamine, δ2.28, 36%;1-dimethylamino-1-oxo-2-hydroxydiazene, sodium salt, 8 2.79, 15%;N-nitrosodimethylamine, δ3.11 and 3.91, 8%; N-nitrososarcosine, sodiumsalt, δ3.15 (s, E methyl), 3.84 (s, Z methyl), 4.21 (s, Z methylene),4.80 (s, E methylene), 10%. The title compound was present as 32% of themixture: δ3.11 (s, 3 H) and 3.60 (s, 2 H).

Example III

[0063] This example illustrates the preparation of1-hydroxy-2-oxo-3-carboxymethyl-3-methyl-1triazene N-methylamide, sodiumsalt, as shown schematically as follows:

[0064] A solution of 150 ml (1.9 mol) of 40% aqueous methylamine wascooled to 0° C. To the solution was added 40 ml of 10 M sodium hydroxidefollowed by the careful addition over a 2-h period at 0° C. ofα-chloroacetyl chloride (27 g, 0.24 mol).

[0065] Stirring was continued at room temperature overnight. Theresulting solution was saturated with sodium chloride and extracted withdichloromethane, dried over sodium sulfate, and filtered through a layerof magnesium sulfate. Most of the solvent was removed on a rotaryevaporator and the residue was distilled at 1 atm then under moderatevacuum. The product distilled at 90-2° C. at 125 mm Hg to yield 15 g(61%).of sarcosine N-methylamide: IR (film) 3318,, 2952, 2889, 1659,1553, 1462, 1413, 1166 cm⁻¹; NMR (CDCl₃) δ2.42 (s, 3 H), 2.86 s 1.5 H),2.83 (s, 1.5 H), 3.23 (s, 2 H).

[0066] A solution of 1.7 g (0.0167 mol) of sarcosine N-methylamide in3.5 ml (0.016 mol) of 25% sodium methoxide in methanol was placed in apressure bottle, flushed with nitrogen and charged with 40 psi of nitricoxide. The solution was kept at 25° C. for 48 h, giving a thick paste.The pressure was released. The residue was washed with ether and driedunder vacuum to give 1.4 g of a solid: UVλ_(max) (ε)250 nm (2.4 mM¹cm⁻¹).

Example IV

[0067] This example illustrates the preparation of the bis(nitric oxide)adduct of L-prolyl-L-leucylglycinamide, as shown schematically asfollows:

[0068] To a slurry of 120 mg (0.423 mmol) ofL-prolyl-L-leucylglycinamide (Sigma) in 4 ml of acetonitrile was added100 μl of 25% sodium methoxide in methanol. The resulting gel wastreated with a few drops of methanol until a homogeneous solution wasobtained. The solution was transferred into a micro-Parr bottle andbubbled with nitrogen for 5 min, followed by exposure to 40 psi of NOfor 72 h. The reaction mixture was dried under vacuum to give 187 mg ofa solid: λ_(max) (ε) 250 nm (6.2 mM⁻¹cm⁻¹) in pH 7.4 buffer. It released0.86 moles of NO (per mole of tripeptide decomposed at this pH) with ahalf-life of 7 min at 37° C.

[0069] Oligopeptides and proteins of increasing chain length can besimilarly derivatized with NO.

Example V

[0070] This example demonstrates the attachment of a nucleophilic centerto a protein that does not contain a nucleophilic center that willreadily react with NO, shown schematically as follows:

[0071] A solution of 4.78 g (0.025 mol) of 17-acetyl-L-methionine inCH₂Cl₂: acetonitrile (120 ml) was cooled to 0° C. To this solution wasadded 5.36 g (0.025 mol) of dicyclohexylcarbodiimide (DCC) followed bythe rapid addition of 3.90 g (0.021 mol) of N-t-butoxycarbonylpiperazinein 6 ml of dichioromethane. The progress of the reaction was followed onsilica gel TLC plates developed with 4:1 acetonitrile: tetrahydrofuranand visualized with either iodine or ninhydrin spray. The reaction wascomplete within 2 h. A few drops of glacial acetic acid were added tothe reaction mixture and the solvent was removed on a rotary evaporator.The residue was taken up in ether and filtered. The clear filtrate waswashed with dilute acid followed by dilute base. The organic layer wasseparated, dried over anhydrous sodium sulfate, filtered, and evaporatedto give 8.2 g of 1-(t-butoxycarbonyl)-4-(N-acetyl-L-methionyl)piperazine, a colorless oil which required nofurther purification: IR (film) 3304, 3058, 2973, 2931, 2868, 1701,1645, 1539, 1420, 1237, 1173 cm⁻¹; NMR (CDCl₃) δ1.47 (.s, 9 H), 1.80 (m,2 H), 2.02 (s, 3 H), 2.10 (s, 3 H), 2.46 (m, 2 H), 3.53 (m, 8 H), 5.10(M, 1 H), 6.35 (b, 0.5 H), 6.43 (b, 0.5 H).

[0072] To a solution of 8.6 g (0.024 mol) of 1-(t-butoxycarbonyl)-4-(N-acetyl-L-methionyl)piperazine in 60 ml of dichloromethane wasadded 10 ml of trifluoroacetic acid and the mixture was stirred at roomtemperature overnight. The solution was extracted with water and theresulting aqueous solution was made basic with sodium hydroxide. Theproduct was extracted with dichloromethane, dried over sodium sulfate,and filtered. Evaporation of the solvent gave 2.1 g of1-(N-acetyl-L-methionyl) piperazine, as an oil: IR (film) 3304, 3051,2917, 2861, 1645, 1546, 1448, 1377 cm⁻¹; NMR (CDCl₃) δ1.95 (m, 2 H),2.02 (s, 3 H), 2.10 (s, 3 H), 2.54 (m, 2 H), 2.98 (m, 4 H), 3.74 (m, 4H), 5.10 (m, 1 H), 6.40 (b, 0.5 H), 6.48 (b, 0.5 H).

[0073] To a solution of 510 mg (1.97 mmol) of 1-(N-acetyl-L-methionyl)piperazine in 1 ml of methanol was added 428 μl (1.97 mmol) of 25%sodium methoxide in methanol. The system was degassed and charged with40 psi of nitric oxide. After exposure of the solution to NO for 120 h,the pressure was released and the solid product was collected byfiltration, washed with ether, and dried to give 27 mg of1-[4-(N-acetyl-L-methionyl)piperazin-l-yl]-1-oxo-2-hydroxydiazene,sodium salt, as a white solid: UV λ_(max) (ε) 252 nm (12.0 mM⁻¹ cm⁻¹).The product decomposed with a half-life of 6.9 min at pH 7 and 25° C. toproduce 1.72 moles of NO per mole of test agent.

Example VI

[0074] This example demonstrates the attachment of a preformed NONOatecontaining a nucleophilic nitrogen atom to the C-terminus of a peptide,polypeptide or protein as shown schematically as follows:

[0075] A solution of 20 g (0.126 mol) of ethyl 1-piperazinecarboxylatein 60 ml of methanol was placed in a Parr bottle. The solution wastreated with 27.4 ml (0.126 mol) of 25% sodium methoxide in methanol.The system was evacuated, charged with 40 psi of nitric oxide and keptat 25° C. for 48 h. The white crystalline product was collected byfiltration and washed with cold methanol as well as with copious amountsof ether. The product was dried under vacuum to give a 14.5 g (48%)yield of 1-(4-carbethoxypiperazin-1-yl)-1-oxo-2-hydroxydiazene, sodiumsalt: mp 184-5° C.; UV (0.01 M NaOH) λ_(max) (ε) 252 nm (10.4 mM⁻¹cm⁻¹); NMR (D₂O) δ1.25 (t, 3 H), 3.11 (m, 2 H), 3.68 (m, 2 H), 4.15 (q,2 H). Anal Calcd. for C₆H₁₃N₄O₄Na: C, 35.00%; H, 5.42%; N, 23.33%; Na,9.58%. Found: C, 34.87%; H, 5.53%; N, 23.26%; Na, 9.69%. The half-lifeof this compound at pH 7 and 25° C. was 5 min. This measurement wasbased on the loss of the 252-nm chromophore in the ultraviolet spectrum.

[0076] A solution of 1.3 g (5.4 mmol) of1-(4-carbethoxypiperazin-1-oxo-2-hydroxydiazene, sodium salt, in 10 mlof 0.01 M aqueous sodium hydroxide was cooled in an ice bath. A solutionof 2 ml of dimethyl sulfate in 10 ml of methanol was added dropwise. Theresulting solution was stirred at 0° C. for 1 h, then allowed to warmgradually to room temperature. After 24 h the solution was concentratedon a rotary evaporator. The residue was extracted with dichloromethane,dried over sodium sulfate, and filtered through a layer of magnesiumsulfate. The solvent was evaporated under reduced pressure and theresidue was chromatographed on silica gel. Elution with 2:1dichloromethane:ethyl acetate provided 683 mg (55%) of1-(4-carbethoxypiperazin-1-y1) -1-oxo-2-methoxydiazene as an oil, whichcrystallized on standing: mp 46° C.; UV λ_(max) (ε) 240 nm (8.4 mM⁻¹cm⁻¹); IR (film) 2988, 2945, 2875, 1707, 1504, 1068 cm⁻¹; NMR δ3.38 (m,4 H), 3.67 (m, 4 H), 4.03 (s, 3 H), 4.16 (q, 2 H); MS m/z (relativeintensity, %), 232 (M⁺, 3), 217 (16), 187 (10), 157 (100), 142 (5), 98(4), 85 (27), 70 (26), 56 (94), 54 (19); exact mass calcd for C₈H₁₆N₄O₄(M⁺) 232.1171, found 232.1172. Anal Calcd for C₈H₁₆N₄O₄: C, 41.38%; H,6.90%; N, 24.14%. Found C, 41.23%; H, 6.82%; N, 24.05%.

[0077] A mixture of 1.8 g (0.0078 mol) of 1-(4-carbethoxypiperazin-1-yl)-1-oxo-2-methoxydiazene and 20 ml of 5 M aqueous sodium hydroxidewas heated at reflux. After 45 min no starting material remained in themixture, as assessed from qualitative thin layer chromatography. Thesolution was allowed to cool to room temperature and evaporated to aviscous residue, which was extracted with ethyl acetate, dried oversodium sulfate, filtered, and evaporated. The product waschromatographed on silica gel and eluted with 1:1dichloromethane:acetone giving 820 mg (66%) of 1-(piperazin-1-yl)-1-oxo-2-methoxydiazene as a pale yellow oil: UV λ_(max) (ε) 234 nm (7.0mM⁻¹ cm⁻¹); NMR δ3.03 (m, 4 H), 3.38 (m, 4 H), 4.06 (s, 3 H); IR (film)3318, 2945, 2854, 1447, 1364, 1286, 1230, 1046, 1004 cm⁻¹; MS m/z(relative intensity, %) 160 (M⁺, 2), 145 (7), 143 (10), 115 (9), 85(56), 58 (7), 56 (100); exact mass calcd for C₅H₁₂N₄O₂ (M⁺) 160.0960,found 160.0966.

[0078] To a solution of 164 mg (0.856 mmol) of N-acetyl-L-methionine in10 ml of 1:1 dichloromethane:acetonitrile was added 206 mg (1 mmol) ofdicyclohexylcarbodiimide (DCC) followed by the rapid introduction of 137mg (0.856. mmol) of 1-(piperazin-1-yl)-1-oxo-2-methoxydiazene in 3 ml ofdichloromethane. The reaction mixture was stirred at 25° C. for 4 h. Afew drops of glacial acetic acid were added to decompose excess DCC. Themixture was filtered and evaporated. The residue was extracted withethyl acetate, which in turn was washed with dilute hydrochloric acid,followed by dilute aqueous sodium hydroxide. The organic layer was driedover sodium sulfate, filtered through, a layer of magnesium sulfate, andevaporated in vacuo. Purification of1-(4-[N-acetyl]-L-methionylpiperazin-1-yl) -1-oxo-2-methoxydiazene wasaccomplished on silica gel using 4:1 acetonitrile:tetrahydrofuran as theeluant: UV λ_(max) (ε) 230 nm (8.7 mM⁻¹ cm⁻¹); NMR δ2.02 (s, 3 H), 2.07(m, 2 H), 2.11 (s, 3 H), 3.46 (m, 4 H), 3.83 (m, 4 H), 4.03 (s, 3 H),5.15 (m, 1 H), 6.28 (b, 0.5 H), 6.35 (b, 0.5 H); IR 3297, 2931, 2847,1645, 1546, 1497, 1441, 1223 cm⁻¹; MS m/z (relative intensity, %), 333(M⁺, 4), 318 (2), 304 (3), 303 (16), 288 (12), 260 (11), 259 (100), 258(9), 214 (78), 184 (37), 183 (10), 174 (5), 146 (26), 142 (56), 141 (5),104 (63), 61 (60); exact mass calcd for C₁₂H₂₃N₅O₄S (M⁺) 333.1470, found333.1471.

[0079] All publications, patents, and patent applications cited-hereinare hereby incorporated by reference to the same extent as if eachindividual document were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein.

[0080] While this invention has been described with emphasis uponpreferred embodiments, it will be obvious to those of ordinary skill inthe art that the preferred embodiments may be varied. It is intendedthat the invention may be practiced otherwise than as specificallydescribed herein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the appended claims.

We claim:
 1. A polymeric composition capable of releasing nitric oxideunder physiological conditions, said composition comprising a biopolymerand a nitric oxide-releasing N₂O₂ ⁻ functional group bound to saidbiopolymer.
 2. The polymeric composition of claim 1, wherein saidbiopolymer is selected from the group consisting of a peptide,polypeptide, protein, oligonucleotide, and nucleic acid.
 3. Thepolymeric composition of claim 2, wherein said biopolymer is selectedfrom the group consisting of a tissue-, cell-, or tumor-specificantibody or fragment thereof, a protein containing a recognitionsequence for a receptor-ligand interaction favorable to tumor cellattachment, an anti-chemotactic agent, and a hormone.
 4. The polymericcomposition of claim 1, wherein said nitric oxide-releasing N₂O₂ ⁻ groupis of the formula

wherein X is an organic or inorganic moiety and X′ is selected from thegroup consisting of X, a pharmaceutically acceptable metal center or apharmaceutically acceptable cation, and wherein said N₂O₂ ⁻ group isbonded to said biopolymer through at least one of X or X′.
 5. Thepolymeric composition of claim 4, wherein said nitric oxide-releasingN₂O₂ ⁻ functional group is of the formula:

wherein J is an organic or inorganic moiety, M^(+x) is apharmaceutically acceptable cation, where x is the valence of thecation, a an integer of at least one, and b and c are the smallestintegers that result in a neutral compound.
 6. The method of claim 5,wherein J is a moiety which is linked to the nitrogen of the remainderof the complex through an atom other than a carbon atom.
 7. Thepolymeric composition of claim 5, wherein the nitric-oxide releasinggroup is a compound other than a salt of alanosine or dopastin.
 8. Thepolymeric composition of claim 4, wherein said nitric oxide-releasingN₂O₂ ⁻ functional group is of the formula:

wherein b and d are the same or different and may be zero or one, R₁,R₂, R₃, R₄, and R₅ are the same or different and may be hydrogen, C₃₋₈cycloalkyl, C₁₋₁₂ straight or branched chain alkyl, benzyl, benzoyl,phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl, or2,2,2-trichloro-t-butoxycarbonyl, and x, y, and z are the same ordifferent and are integers from 2 to
 12. 9. The polymeric composition ofclaim 4, wherein said nitric oxide-releasing N₂O₂ ⁻ functional group isof the formula:

and R₇ are the same or different and may be hydrogen, C₃₋₈ cycloalkyl,c₁₋₁₂ straight or branched chain alkyl, benzyl, benzoyl, phthaloyl,acetyl, trifluoroacetyl, p-toyuyl, t-butoxycarbonyl, or2,2,2-trichloro-t-butoxycarbonyl, f is an integer from 0 to 12, with theproviso that when B is the substituted piperazine moiety

then f is an integer from 2 to
 12. 10. The polymeric composition ofclaim 4, wherein said nitric oxide-releasing N₂O₂ ⁻ functional group isof the formula:

wherein R₈ is hydrogen, C₃₋₈ cycloalkyl, C₁₋₁₂ straight or branchedchain alkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl,p-toluyl, t-butoxycarbonyl, or 2,2,2-tri-chloro-t-butoxycarbonyl, R₉ ishydrogen or a c₁-C₁₂ straight or branched chain alkyl, and g is 2 to 6.11. The polymeric composition of claim 4, wherein said nitricoxide-releasing N₂O₂ ⁻ functional group is of the formula:

wherein R₁ and R₂ are independently selected from the group consistingof a straight chain or branched chain C₁-C₁₂ alkyl group and a benzylgroup, or else R₁ and R₂ together with the nitrogen atom they are bondedto form a heterocyclic group, a pyrrolidino, piperidino, piperazino ormorpholino group, M^(+x) is a pharmaceutically acceptable cation, and xis the valence of the cation.
 12. The polymeric composition of claim 4,wherein said nitric oxide-releasing N₂O₂ ⁻functional group is of theformula: K[(M)^(x′) _(x)(L)_(y)(R¹R²N—N₂O₂)_(z)]  (VI) wherein M is apharmaceutically acceptable metal, or, where x is at least two, amixture of two different pharmaceutically acceptable metals, L is aligand different from (R¹R²N—N₂O₂) and is bound to at least one metal,R¹ and R² are each organic moieties and may be the same or different, xis an integer of from 1 to 10, x′ is the formal oxidation state of themetal M, and is an integer of from 1 to 6, y is an integer of from 1 to18, and where y is at least 2, the ligands L may be the same ordifferent, z is an integer of from 1 to 20, and K is a pharmaceuticallyacceptable counterion to render the compound neutral to the extentnecessary.
 13. The polymeric composition of claim 4, wherein said nitricoxide-releasing N₂O₂ ⁻ functional group is of the formula:[R—N(H)N(NO)O—]_(y)X  (VII) wherein R is C₂₋₈ lower alkyl, phenyl,benzyl, or C₃₋₈ cycoloalkyl, any of which R groups may be substituted byone to three substituents, which are the same or different, selectedfrom the group consisting of halo, hydroxy, C₁₋₈ alkoxy, —NH₂, —C(O)NH₂,—CH(O), —C(O) OH, and —NO₂, X is a pharmaceutically acceptable cation, apharmaceutically acceptable metal center, or a pharmaceuticallyacceptable organic group selected from the group consisting of C₁₋₈lower alkyl, —C(o)CH₃, and —C(O)NH₂, and y is one to three, consistentwith the valence of X.
 14. The polymeric composition of claim 4, whereinsaid nitric oxide-releasing N₂O₂ ⁻ functional group is of the formula:

wherein R₁ and R₂ are independently chosen from C₁₋₁₂ straight chainalkyl, C₁₋₁₂ alkoxy or acyloxy substituted straight chain alkyl, C₂₋₁₂hydroxy or halo substituted straight chain alkyl, C₃₋₁₂ branched chainalkyl, C₃₋₁₂ hydroxy, halo, alkoxy, or acyloxy substituted branchedchain alkyl, C₃₋₁₂ straight chain olefinic and C₃₋₁₂ branched chainolefinic which are unsubstituted or substituted with hydroxy, alkoxy,acyloxy, halo or benzyl, or R₁ and R₂ together with the nitrogen atom towhich they are bonded form a heterocyclic group, a pyrrolidino,piperidino, piperazino or morpholino group, and R₃ is a group selectedfrom C₁₋₁₂ straight chain and C₃₋₁₂ branched chain alkyl which areunsubstituted or substituted by hydroxy, halo, acyloxy or alkoxy, C₂₋₁₂straight chain or C₃₋₁₂ branched chain olefinic which are unsubstitutedor substituted by halo, alkoxy, acyloxy or hydroxy, C₁₋₁₂ unsubstitutedor substituted acyl, sulfonyl and carboxamido; or R₃ is a group of theformula —(CH₂)_(n)—ON═N(O)NR₁R₂, wherein n is an integer of 2-8, and R₁and R₂ are as defined above; with the proviso that R₁, R₂ and R₃ do notcontain a halo or a hydroxy substituent α to a heteroatom.
 15. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and the polymeric composition of claim
 1. 16. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and thepolymeric composition of claim
 2. 17. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and the polymericcomposition of claim
 3. 18. A pharmaceutical composition comprising apharmaceutically acceptable carrier and the polymeric composition ofclaim
 4. 19. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and the polymeric composition of claim
 5. 20. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and the polymeric composition of claim
 6. 21. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and thepolymeric composition of claim
 7. 22. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and the polymericcomposition of claim
 8. 23. A pharmaceutical composition comprising apharmaceutically acceptable carrier and the polymeric composition ofclaim
 9. 24. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and the polymeric composition of claim
 10. 25. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and the polymeric composition of claim
 11. 26. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and thepolymeric composition of claim
 12. 27. A method of treating a biologicaldisorder in a mammal in which dosage with nitric oxide is therapeutic,comprising administering to said mammal the polymeric composition ofclaim 1 in an amount sufficient to release a therapeutically effectiveamount of nitric oxide.
 28. A method of treating a biological disorderin a mammal in which dosage with nitric oxide is therapeutic, comprisingadministering the polymeric composition of claim 2 in an amountsufficient to release a therapeutically effective amount of nitricoxide.
 29. A method of treating a biological disorder in a mammal inwhich dosage with nitric oxide is therapeutic, comprising administeringthe polymeric composition of claim 3 in an amount sufficient to releasea therapeutically effective amount of nitric oxide.
 30. A method oftreating a biological disorder in a mammal in which dosage with nitricoxide is therapeutic, comprising administering the polymeric compositionof claim 4 in an amount sufficient to release a therapeuticallyeffective amount of nitric oxide.
 31. A method of treating a biologicaldisorder in a mammal in which dosage with nitric oxide is therapeutic,comprising administering the polymeric composition of claim 5 in anamount sufficient to release a therapeutically effective amount ofnitric oxide.
 32. A method of treating a biological disorder in a mammalin which dosage with nitric oxide is therapeutic, comprisingadministering the polymeric composition of claim 6 in an amountsufficient to release a therapeutically effective amount of nitricoxide.
 33. A method of treating a biological disorder in a mammal inwhich dosage with nitric oxide is therapeutic, comprising administeringthe polymeric composition of claim 7 in an amount sufficient to releasea therapeutically effective amount of nitric oxide.
 34. A method oftreating a biological disorder in a mammal in which dosage with nitricoxide is therapeutic, comprising administering the polymeric compositionof claim 8 in an amount sufficient to release a therapeuticallyeffective amount of nitric oxide.
 35. A method of treating a biologicaldisorder in a mammal in which dosage with nitric oxide is therapeutic,comprising administering the polymeric composition of claim 9 in anamount sufficient to release a therapeutically effective amount ofnitric oxide.
 36. A method of treating a biological disorder in a mammalin which dosage with nitric oxide is therapeutic, comprisingadministering the polymeric composition of claim 10 in an amountsufficient to release a therapeutically effective amount of nitricoxide.
 37. A method of treating a biological disorder in a mammal inwhich dosage with nitric oxide is therapeutic, comprising administeringthe polymeric composition of claim 11 in an amount sufficient to releasea therapeutically effective amount of nitric oxide.
 38. A method oftreating a biological disorder in a mammal in which dosage with nitricoxide is therapeutic, comprising administering the polymeric compositionof claim 12 in an amount sufficient to release a therapeuticallyeffective amount of nitric oxide.